Impact of Putrescine on essential oil composition, free polyamines content and expression of related genes (DXR and TPS2) in Thymus daenensis under drought stress

Impact of Putrescine on essential oil composition, free polyamines content and expression of related genes (DXR and TPS2) in Thymus daenensis under drought stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of Putrescine on essential oil composition, free polyamines content and expression of related genes (DXR and TPS2) in Thymus daenensis under drought stress Ensiyeh Shahroudi, Fatemeh Zarinkamar, Bahram M. soltani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6523120/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in BMC Plant Biology → Version 1 posted 16 You are reading this latest preprint version Abstract Background Thymus daenensis a medicinal plant native to Iran, produces essential oils rich in thymol and carvacrol, known for their strong antioxidant and antimicrobial properties. Exposure to drought conditions affects the production pathways of secondary metabolites, potentially contributing to improved stress tolerance in plants. Polyamines (PAs), particularly putrescine (Put), play a crucial role in mitigating drought stress by regulating stomatal behavior, improving osmotic adjustment, and modulating oxidative stress responses. This study aimed to investigate the effects of exogenous Put on T. daenensis under drought stress, focusing on gene expression related to terpenoid biosynthesis, secondary metabolite accumulation, and free PA content. Results Six-week-old T. daenensis plants were subjected to drought stress induced by 15% (w/v) polyethylene glycol (PEG) 6000, with or without foliar application of Put. Gene expression analysis (RT-qPCR), HPLC-based PA quantification, and GC-MS profiling of essential oil composition were performed. Put treatment significantly increased endogenous Put and spermidine content in drought-stressed plants, whereas no significant changes were observed in non-stressed plants. Furthermore, Put application upregulated TPS2 but downregulated DXR expression in drought-stressed plants. Additionally, the relative contents of γ -terpinene and p -cymene—precursors of thymol and carvacrol—increased, while the contents of thymol and carvacrol decreased following Put treatment. Conclusions Exogenous application of putrescine modulates the expression of key genes in the terpenoid biosynthetic pathway and alters essential oil composition in T. daenensis under drought stress, potentially contributing to the plant’s adaptive responses. Drought stress Gene expression (DXR TPS2) Putrescine Terpenoid biosynthesis Thymus daenensis Figures Figure 1 Background T. daenensis is a species of thyme native to Iran ( 1 ). It's known for its medicinal properties and is widely used in traditional medicine ( 2 ). The essential oil of T. daenensis is rich in compounds like thymol, carvacrol, p -cymene, and β -caryophyllene, which have antioxidant, antibacterial, and antifungal properties ( 1 ). Thymol and carvacrol exhibit strong antioxidant properties, helping to neutralize reactive oxygen species (ROS) generated during stresses. These compounds reduce oxidative damage to cellular components by stabilizing cell membranes, protecting them from damage in relation to drought-induced dehydration ( 3 ). Thymol and carvacrol can influence stomatal behavior, promoting stomatal closure to reduce water loss and improve water-use efficiency ( 3 ). Drought stress can lead to increase in the production of some secondary metabolites. This enhanced production helps the plant cope with stress and defend against pathogens ( 4 ). Thymol and carvacrol are phenolic monoterpenes, and their production is primarily driven by the methyl erythritol phosphate (MEP) pathway ( 5 ). The pathway begins with the generation of dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IPP), which are key building blocks for terpenes biosynthesis ( 5 ). DMADP and IPP are converted into 1-deoxy-D-xylulose-5-phosphate (DXP) through a series of enzymatic reactions. DXP is then transformed into 2C-methyl-D-erythritol-4-phosphate (MEP) via 1-deoxy-D-xylulose-5-phosphate reductoisomerase ( DXR ) ( 5 ). MEP is further processed to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMADP), which are used to synthesize monoterpenes ( 5 ). IPP and DMADP are converted into geranyl diphosphate (GPP), which is then cyclized and oxidized to form monoterpene compounds such as thymol and carvacrol by Gamma-terpinene synthase ( TPS2 ) ( 6 ). The enzymes involved in this pathway, such as DXR and TPS2 , play crucial roles in the monoterpene biosynthesis ( 7 ). PAs are small aliphatic nitrogen-containing compounds characterized by the presence of two or more amino groups ( 8 ). These molecules are widely distributed across all living organisms, including both eukaryotic and prokaryotic cells ( 9 ). The most prevalent forms of PAs include the diamine Put, the triamine spermidine (Spd), and the tetraamine spermine (Spm) ( 10 , 11 ). Put is considered the central product in the biosynthetic pathway of PAs, serving as the primary precursor for the synthesis of both Spd and Spm ( 12 ). Several studies have indicated that the application of exogenous PAs can improve plant resistance to drought stress ( 13 , 14 ). These compounds have been found to stimulate the production of secondary metabolites, including alkaloids, phenolic compounds, and terpenoids, which are essential for plant defense and overall health ( 15 ). Additionally, it has been shown that optimal foliar application of Put can activate key physiological processes, promoting the synthesis of osmotic regulators such as free amino acids, soluble sugars, and proline. This mechanism may help mitigate the negative impacts of drought on plant biomass, thus improving both the quality and quantity of specific bioactive compounds ( 13 , 16 ). PAs play a dual role in regulating oxidative stress in plants ( 17 ). On one hand, they enhance the activity of various antioxidant enzymes, thereby helping to mitigate oxidative stress induced by environmental factors ( 18 ). On the other hand, PAs can serve as a source of reactive oxygen species (ROS), as their catabolism generates potent oxidants such as hydrogen peroxide (H 2 O 2 ) and acrolein, which may contribute to cellular damage under stress conditions. However, H 2 O 2 also functions as a signaling molecule, initiating the stress signal transduction cascade and activating antioxidant defense mechanisms. Consequently, PAs act as key regulators of redox homeostasis, balancing both protective and potentially harmful effects in response to oxidative stress. We hypothesized that the application of Put alleviates drought stress in T. daenensis by enhancing the biosynthesis and accumulation of some compounds present in the essential oil, regulating the expression of key biosynthetic genes ( DXR and TPS2 ) and increasing PAs content. This effect is expected to occur through the modulation of stress-responsive metabolic and genetic pathways, leading to improved drought tolerance and enhanced secondary metabolite production in the plant. In essence, our hypothesis suggests that Put plays a protective and stimulatory role in plants under drought conditions, likely involving the coordinated regulation of secondary metabolite biosynthesis, PA accumulation, and specific gene expression. Materials and methods Plant materials and treatments To conduct this study, T. daenensis seeds were sourced from the Isfahan Agricultural and Natural Resources Research and Educational Center in Iran. The seeds were sown in individual plastic pots filled with perlite. The pots were kept in a phytotron chamber under light conditions of 16 hours of light and 8 hours of darkness, with a temperature of 25°C and light intensity of 1200–1400 lux (17–20 µmol photons/m²/s) for six weeks. During this time, each pot was irrigated uniformly with a half-strength Hoagland solution. After this initial growth period, drought stress was induced using a polyethylene glycol (PEG) solution (15% W/V), applied with and without Put at a concentration of 0.2 mM as described by ( 13 ). 100 mL of the PEG-containing half-strength Hoagland solution was added to each pot, while 10 mL of Put solution was uniformly sprayed onto the plants using an atomizer on alternate days. Each experimental treatment was performed in triplicate to ensure data reliability. At the end of the three-week treatment period, shoots were collected from each condition and a portion of the them were dried, while the remaining ones were stored − 80°C. Total RNA extraction and cDNA synthesis Total RNA was isolated utilizing the Riboex reagent according to the protocol provided by the manufacturer. The concentration and purity of the extracted RNA were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and verified by agarose gel electrophoresis. Samples exhibiting an A260/A280 ratio between 1.8 and 2.1 and an A260/A230 ratio above 1.7 were selected for downstream complementary DNA (cDNA) synthesis. cDNA was synthesized from the purified RNA using reverse transcription kits obtained from Thermo Fisher (USA), adhering strictly to the provided instructions. Quantitative real-time PCR (RT-qPCR) was performed to evaluate the expression of DXR and TPS2 genes, employing SYBR Green PCR Master Mix (2×) (Yekta Tajhiz Azma, Tehran, Iran). The amplification protocol involved an initial denaturation at 95°C for 5 minutes, followed by 40 cycles comprising 10 seconds at 95°C for denaturation, 10 seconds at 60°C for annealing, and 15 seconds at 72°C for extension. Reactions were run on an Applied Biosystems® StepOne™ instrument (Thermo Fisher, USA), and data analysis was conducted using the StepOne Software v2.3. Gene expression levels were normalized to GAPDH and analyzed using the 2^–ΔΔCt method described by Livak and Schmittgen ( 19 ). All assays were carried out with two independent biological replicates and two technical replicates per sample. Primers designing For primer designing, the sequence of target genes was first examined on the NCBI website (gov.nih.nlm.ncbi.www). The sequence for the Gamma terpinene synthase ( TPS2 ) gene was found for the T. daenensis species. However, no registered sequences for the GAPDH and DXR genes of this species were available. Therefore, gene sequences from a closely related species, T. vulgaris , were used. The accuracy of these sequences was confirmed by comparison with the gene bank, and primers for Real-Time PCR (RT-qPCR) were then designed based on these sequences (Table 1 ). Table 1 Specifications of primers used for RT-qPCR (Real time PCR) Gene Sequences Amplicone size (bp) Accession no. GAPDH Forward:5ʹGTGCTTCCAGCTTTGAACG-3ʹ Riverse: 5ʹGTTCTCTGACTCCTCCTTGATG-3ʹ 147 >MF373628.1 DXR Forward: 5ʹ-GTGCTAGCTCAGTTAGGA TGG-3ʹ Riverse: 5ʹ-TTAGATCGACGTTGCAGAGG 124 >KY621335.1 TPS2 Forward: 5ʹ-CTTACAAGGCGAGGA AGG ACA-3ʹ Riverse: 5ʹ-CACAAATGGGAGTTTCTCGG-3ʹ 149 >MH686193.1 Measurement of PA content using HPLC PAs were extracted following the method of Sharma & Rajam (1995). 0.2 g of samples was homogenized with 1 mL of 2% perchloric acid (PCA) and centrifuged at 4°C for 20 minutes. Then, 150 µL of the supernatant was mixed with 200 µL of saturated sodium carbonate and 500 µL of dansyl chloride solution (5 mg/mL) for dansylation. The mixture was incubated at 60°C in the dark for 1 hour. Afterward, 200 µL of proline solution (0.1 g/mL) was added, and the solution was kept under the same conditions for an additional 30 minutes. The dansylated PAs were extracted by vortexing with 500 µL of toluene, and the upper phase containing the PAs was collected for subsequent analysis. PAs quantification was performed using an ODS18-C5 column. The mobile phase consisted of acetonitrile and acidified water (deionized water with 0.01% acetic acid) applied using a standard gradient system. The column used was an ODS18-C3 type with a length of 250 mm and a diameter of 4.6 mm. PAs absorption was detected using a UV detector at a wavelength of 254 nm. The quantification of PAs was performed based on the chromatogram, using their respective standard curves ( 21 ). GC.MS 20 mg portion of powdered plant dry tissue was placed in 20 mL vials. Static headspace analysis was performed using an A7697 Headspace Sampler (Agilent Technologies). The headspace oven temperature was maintained at 80°C for 30 minutes to allow volatile compounds to evaporate and reach equilibrium between the headspace and the sample in the vial. Subsequently, a specialized syringe was used to extract 1 mL of the headspace, which was then injected into a gas chromatography-mass spectrometry (GC-MS) system for analysis. An Agilent 7890 gas chromatograph, coupled with an HP-5MS column (30 m length, 0.25 mm internal diameter, and 250 µm film thickness), and an ELCD 5320 detector, was employed for the analysis. The temperature program for the oven was as follows: an initial ramp from 60°C to 210°C at a rate of 3°C/min, followed by a temperature increase to 240°C at 20°C/min, and a subsequent hold at 240°C for 8.5 minutes. The total run time was 60 minutes, with the electron ionization energy set to 70 eV. Helium was used as the carrier gas with a flow rate of 1 mL/min. Data analysis was conducted using ChemStation software. Statistical analysis All data were analyzed using analysis of variance (ANOVA) based on a completely randomized design (CRD) with three replications. Duncan’s test was applied to determine statistically significant differences between treatment means at a significance level of p ≤ 0.05. Results and discussion Relative Content of Essential Oil Compounds and Expression of DXR and TPS2 Genes : The impact of drought stress on essential oil content in plants varies depending on the plant species, the intensity and duration of stress, and the growth stage ( 22 ). Some studies have shown that drought stress can enhance essential oil content by stimulating the biosynthesis of terpenoids, phenylpropanoids, and other aromatic compounds ( 22 , 23 ). Conversely, other studies have indicated that drought stress can reduce essential oil content by limiting biomass production and the availability of precursors ( 24 ). Thus, the optimal irrigation level for maximizing essential oil content depends on the plant species and environmental conditions ( 23 ). A study on T. daenensis demonstrated that Put increased the content of thymol and other secondary metabolites such as carvacrol, α and γ- terpinene, p -cymene, and β -caryophyllene ( 26 ). Moreover, it increased the content of terpenoids, especially mono- and sesquiterpenes, in tea plants under saline stress ( 24 ). Another study found that Put reduced terpenoid content in thyme under drought stress but increased the phenolic compound content ( 25 ). These findings suggest that Put regulates the biosynthesis of various secondary metabolites in plants, depending on environmental conditions and genetic factors. Generally, the mechanism of PAs effects under drought stress is not fully understood but may involve regulation of enzyme gene expression and their activity, transcription factors, and signaling pathways ( 26 ). In this study, the thymol content increased by 4.4% in drought-stressed plants compared to controls (Table 2 ). Similar results were previously observed in garden thyme ( 26 ) and oregano ( 27 ). Research on T. daenensis subsp. daenensis showed that drought stress (20% field capacity) increased thymol content by 17.5% ( 29 ). Additionally, drought stress (50% field capacity) increased thymol content by 13.6% in the first growth season and 9.8% in the second growth season, compared to controls ( 30 ). In addition to drought stress at 40% field capacity, thymol content increased by 10.6% in the first year and 7.8% in the second year in T. daenensis ( 30 ). These results suggest that drought stress enhances thymol biosynthesis as a defensive mechanism against oxidative stress in T. daenensis . However, the optimal level and duration of drought stress depend on plant genotype, climate, and soil conditions ( 31 ). The ratio of thymol to other compounds in the essential oil affects the quality of cosmetic, food, and pharmaceutical products derived from this plant ( 32 ), Therefore, it can be suggested that the quality of T. daenensis essential oil may improve under drought stress. Table 2 The effect of drought stress and Put on the relative content of compounds in the essential oil of T. daenensis . substance KI RT (min) Relative content of substance (%) Average Control Put PEG Put + PEG (%) Thymol 1290 18.5 41.53 c ± 0.89 45.49 a ± 0.57 43.37 b ± 0.59 36.27 d ± 0.54 41.66 Carvacrol 1298 18.7 1.6 a ± 0.04 1.36 b ± 0.01 1.50 a ± 0.04 1.25 b ± 0.04 1.42 γ -Terpinene 1050 9.2 9.34 d ± 0.06 17.71 a ± 0.11 11.67 c ± 0.29 15.00 b ± 0.20 13.43 p -cymene 1011 8.0 15.60 a ± 0.29 15.18 a ± 0.05 8.87 b ± 0.17 15.87 a ± 0.30 13.88 α -Thujene 905 5.5 1.42 c ± 0.19 2.50 c ± 0.29 5.07 b ± 0.40 5.30 a ± 0.28 3.57 Camphene 943 5.9 0.57 a ± 0.76 nd 0.45 a ±0.58 0.66 a ±0.78 0.44 β -Pinene 970 6.9 0.66 a ±0.07 nd 0.69 a ±0.08 0.69 a ±0.08 0.51 β -Myrcene 980 6.9 0.50 c ±0.05 1.82 b ±0.08 3.49 a ±0.15 nd 1.45 α -Terpinen 1010 7.8 2.50 a ±0.08 1.44 c ±0.04 2.14 b ±0.03 nd 1.52 β -Phellandrene 1030 8.4 0.90 nd nd nd 0.22 α – Phellandrene 1008 7.6 nd nd 0.73 0.91 0.41 Endo-Borneol 1165 13.3 1.00 a ±0.28 1.35 a ±0.08 1.48 a ±0.10 1.14 a ±0.02 1.24 Thymol methyl ether 1235 16.1 0.25 c ±0.05 0.37 bc ±0.04 0.61 a ±0.06 0.46 ab ±0.05 0.42 Carvacrol methyl ether 1244 16.5 3.63 b ±0.28 4.46 a ±0.11 4.88 a ±0.17 4.35 a ±0.11 4.33 Thymoquinone 1249 16.7 1.95 c ±0.11 3.57 a ±0.06 2.94 b ±0.17 0.33 d ±0.05 2.19 Caryophyllene 1414 23.7 4.50 b ±0.08 6.72 a ±0.06 5.73 ab ±0.07 5.20 b ±0.9 5.53 Aromandendrene 1414 24.5 0.14 a ±0.01 0.17 a ±0.01 0.18 a ±0.01 0.19 a ±0.02 0.17 Humulene 1450 25.0 0.08 b ±0.02 0.15 a ±0.02 0.10 b ±0.02 0.09 b ±0.03 0.10 β -Bisabolene 1500 27.3 0.32 b ±0.02 0.57 a ±0.04 0.44 ab ±0.08 0.43 ab ±0.07 0.44 (Z)- α -Bisabolene 1538 28.6 1.07 a ±0.10 1.34 a ±0.19 1.11 a ±0.06 1.39 a ±0.10 1.22 α -Pinene 930 5.9 nd 0.25 b ±0.07 0.93 a ±0.17 0.73 a ±0.10 0.47 Eucalyptol 1023 8.3 nd 1.39 a ±0.16 1.57 a ±0.08 1.63 a ±0.11 1.14 (+)-2-Carene 995 7.8 nd nd nd 2.76 0.69 Unknown - - 8.25 a ±1.0 2.53 bc ±0.17 1.91 c ±0.23 5.22 b ±0.12 3.72 The data are the mean of three replicates ± SE. Different letters show statistically significant differences at p ≤ 0.05 level. Table 3 The effect of drought stress and Put on the relative expression of the DXR and TPS2 gene in T. daenensis . Treatment DXR expression (fold change) TPS2 expression (fold change) Control 1 C 1 C Put (0.2 mM) 1.51 ± 0.16 C 23.31 ± 3.69 a PEG (15%) 13.34 ± 2.61 a 1.52 ± 0.23 C Put + PEG 5.66 ± 1.20 b 4.17 ± 0.76 b The data are the mean of three replicates ± SE. Different letters show statistically significant differences at p ≤ 0.05 level. As shown in Table 2 , Put treatment increased thymol content by 9.5%. However, in plants under drought stress, Put caused a 16.37% reduction in thymol content (Table 2 ). One possible explanation for this reduction is that Put regulates the biosynthesis of various secondary metabolites based on genetic factors and environmental conditions ( 33 ). Consequently, Put may reduce thymol content while increasing the levels of other secondary metabolites that could play a more protective role under stress conditions ( 34 ). Based on our results, drought stress also reduced carvacrol content by 6.25% (Table 2 ). Previous studies reported a decline in carvacrol content in T. daenensis ( 35 ), garden thyme ( 26 , 30 ), and Himalayan thyme ( 22 ) under drought stress. In contrast, it is found that carvacrol content increased under mild drought stress but decreased under severe stress in Satureja hortensis ( 36 ). Under non-stress and stress conditions, Put reduced the carvacrol content by 15% and 16.6%, respectively (Table 2 ). Previous studies reported a positive effect of Put on carvacrol content; for instance, Put increased carvacrol content by 2.10% in oregano. A potential explanation for the reduction in carvacrol content with Put treatment is the conversion of carvacrol to thymoquinone, as Put increased thymoquinone (TQ) content by 83% in non-stressed plants. The content of p -cymene, a precursor for thymol and carvacrol, showed a significant decrease (43.14%) under drought stress (Table 2 ). Similar results were observed in T. daenensis ( 33 ) and Satureja hortensis ( 36 ). This negative effect could be due to the inhibitory role of drought stress on the monoterpene biosynthesis pathway and reduced activity of related enzymes ( 37 ). Also, our results indicated that Put reduced p -cymene content by 2.6% under non-stress conditions while increased it’s content by 56% under drought stress (Table 2 ). The expression of TPS2 , the gene encoding the enzyme involved in γ -terpinene biosynthesis, increased 1.5-fold under drought stress (Table 3 ). Studies have shown that γ -terpinene content increases under mild to moderate drought stress in thyme species ( 36 , 27 ), while it decreases under severe stress, as observed in garden thyme ( 39 ) and T. daenensis ( 33 ). The discrepancy in results may be related to the plant's adaptive mechanisms, such as changes in proline content, potassium levels, and antioxidant activity, in response to drought ( 35 , 33 ). Put treatment increased γ -terpinene content by 89.6%, (Table 2 ) with a 23.3-fold increase in TPS2 gene expression compared to controls (Table 3 ). Under drought stress, Put enhanced γ -terpinene content by 28.5-fold and TPS2 gene expression by 2.7-fold (Table 2 ). TQ, an important compound in the essential oil of T. daenensis , is derived from thymol and carvacrol. Drought stress increased TQ content by 50.7% (Table 2 ). Mild drought stress (40% field capacity) increased TQ content, while severe drought stress (20% field capacity) decreased it ( 27 ). TQ has been suggested to enhance the antioxidant and antimicrobial activities of thyme essential oil. Studies indicate that TQ mitigates drought stress by modulating growth regulators like abscisic acid, salicylic acid, and jasmonic acid ( 38 , 39 ).TQ also boosts the activity of enzymes like superoxide dismutase, catalase, and ascorbate peroxidase ( 42 ), as well as non-enzymatic antioxidants such as ascorbic acid, glutathione, and phenolic compounds. Additionally, TQ improves relative water content (RWC), water use efficiency (WUE), and proline accumulation ( 42 ). Free PAs Content During various stress conditions, the accumulation of PAs contributes to plant survival and stress tolerance. The ability of PAs to mitigate stress effects is attributed to their antioxidant properties ( 43 ) and their role in maintaining cellular turgor pressure ( 32 ). PAs in plants are synthesized through the enzymatic activities of ornithine decarboxylase and arginine decarboxylase. Under stress conditions, the biosynthesis of these compounds is increased through the activity of arginine decarboxylase ( 44 ). Numerous studies have been reported that PAs have a modulating function in plants exposed to drought stress ( 41 ). Osmotic stress induced by mannitol has been shown to increase the levels of Put, Spd, and Spd in wheat plants ( 43 ). Research findings indicate that under drought stress, the activity of ornithine decarboxylase and arginine decarboxylase, as well as the content of PAs, is higher in drought-tolerant species compared to drought-sensitive ones ( 44 ). This underscores the role of PAs in enhancing plant resistance to stress. Drought stress decreased the content of endogenous Put by 26.3% and had no effect on Spd content. A study on barley ( Hordeum vulgare L.) seedlings reported that 21-day drought stress resulted in an 80% reduction in endogenous Put levels. However, the same stress conditions led to increases in Spd (50%) level. Put treatment increased the free Put content (Fig. 1 .a) by 55% and free Spd content (Fig. 1 .b) by 23.1% in plants under drought stress. The application of exogenous putrescine under drought conditions was found to mitigate some of the adverse effects of drought stress ( 47 ). Similarly, researchers observed that drought stress led to a decrease in free Spd levels at all developmental stages in both traditional and semi-dwarf triticale (× Triticosecale Wittm .) cultivars. Additionally, a reduction in free Put was noted in the traditional cultivar under drought conditions ( 48 ). In another study, the effects of osmotic stress induced by PEG on polyamine content in soybean ( Glycine max L.) leaves were examined. The findings indicated that drought-tolerant soybean cultivars exhibited higher increases in free Spd and Spm levels, while the drought-sensitive cultivars showed a higher increase in free putrescine under stress conditions. This suggests a complex relationship between polyamine levels and drought tolerance in soybean ( 49 ). Exogenous application of Put significantly influences the endogenous levels of PAs, including Put itself and Spd, in plants. The effects observed can vary depending on the plant species, developmental stage, and environmental conditions ( 50 ). These changes are mediated through the regulation of genes associated with PA metabolism, highlighting the intricate balance plants maintain in PA homeostasis. Applying Put externally often leads to an increase in the plant's internal Put concentration. For instance, in Picea abies somatic embryos, treatment with exogenous Put at concentrations of 10, 100, or 500 µM resulted in a significant, concentration-dependent rise in endogenous Put levels. This suggests that externally supplied Put can be absorbed and utilized by plant tissues, thereby elevating internal Put ( 50 ). Conversely, research on Vitis vinifera (grapevine) seedlings under drought stress demonstrated that exogenous Put application not only increased endogenous Put content but also influenced Spd levels. Specifically, certain treatments led to a significant rise in Spd content, suggesting that exogenous Put can serve as a precursor for the biosynthesis of higher PAs like Spd, especially under stress conditions ( 51 ). Conclusion This study highlights the significant role of Put in mitigating drought stress in T. daenensis by modulating gene expression, PAs accumulation, and secondary metabolite composition. The findings highlight that exogenous Put application significantly increases endogenous Put and Spd levels under drought conditions, suggesting its involvement in osmotic adjustment and oxidative stress regulation. At the molecular level, Put treatment upregulated TPS2 while downregulating DXR expression in drought-stressed plants, indicating its influence on the terpenoid biosynthetic pathway. These genetic modifications were accompanied by a decrease in thymol and carvacrol content, alongside an increase in their precursors, γ -terpinene and p -cymene. This shift in essential oil composition suggests that Put may regulate metabolic fluxes within the plant’s secondary metabolism in response to drought stress. Overall, these results underscore the potential of PAs, particularly Put, as biostimulants for improving drought tolerance and metabolic regulation in medicinal plants. Further investigations are needed to assess the long-term physiological effects and potential agronomic applications of Put in T. daenensis cultivation under field conditions. Abbreviations DMAPP Dimethylallyl pyrophosphate DXP 1-deoxy-D-xylulose 5-phosphate DXR 1-deoxy-D-xylulose 5-phosphate reductoisomerase GAPDH Glyceraldehyde-3-phosphate dehydrogenase GPP Geranyl pyrophosphate H₂O₂ Hydrogen peroxide IPP Isopentenyl pyrophosphate MEP Methylerythritol phosphate PA Polyamine PEG polyethylene glycol Put Putrescine ROS Reactive oxygen species RT-qPCR Quantitative real-time PCR Spd Spermidine Spm Spermine T . daenensis Thymus daenensis TPS2 Terpene synthase 2 Declarations Acknowledgments I would like to express my sincere gratitude to the University of Tarbiat Modares for providing me with the necessary resources and support throughout the course of this research. Author Contributions Ensiyeh Shahroudi designed and performed the experiments, collected and analyzed the data, and wrote the manuscript. Dr. Fatemeh Zarinkamar supervised the physiological experiments conducted in her laboratory. Dr. Bahram M. Soltani supervised the molecular experiments conducted in his laboratory. Funding The authors received no external funding for this work. Ethics approval and consent to participate This study did not involve any human participants or animals. Thymus daenensis seeds were sourced from the Isfahan Agricultural and Natural Resources Research and Educational Center (Iran), and the plants were cultivated and used in accordance with institutional, national, and international guidelines. Therefore, no ethical approval or consent to participate was required. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Alizadeh A, Alizadeh O, Amari G, Zare M. Essential oil composition, total phenolic content, antioxidant activity and antifungal properties of Iranian Thymus daenensis subsp. daenensis Celak. as in influenced by ontogenetical variation. J Essent Oil Bear Plants. 2013;16(1):59–70. 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Aziz EE, Hendawy SF. Effect of soil type and irrigation intervals on plant growth, essential oil yield and constituents of Thymus vulgaris plant. 2008. Said-Al AHAH, Omer EA, Naguib NY. Effect of water stress and nitrogen fertilizer on herb and essential oil of oregano. Int Agrophysics. 2009;23(3):269–75. Kachoie MA, Pirbalouti AG, Hamedi B, Borojeni HAR, Pishkar GR. Effect of drought stress on antibacterial activity of Thymus daenensis subsp. daenensis Celak. J Med Plants Res. 2013;7(36):2923–7. Bistgani ZE, Siadat SA, Bakhshandeh A, Pirbalouti AG, Hashemi M. Interactive effects of drought stress and chitosan application on physiological characteristics and essential oil yield of Thymus daenensis Celak. Crop J. 2017;5(5):407–15. Schweiger AH, Zimmermann T, Poll C, Marhan S, Leyrer V, Berauer BJ. The need to decipher plant drought stress along the soil–plant–atmosphere continuum. Oikos. 2023;2023(9):e10136. Letchamo W, Xu HL, Gosselin A. Photosynthetic potential of Thymus vulgaris selections under two light regimes and three soil water levels. Sci Hortic (Amsterdam). 1995;62(1–2):89–101. Ma Y, Dias MC, Freitas H. Drought and salinity stress responses and microbe-induced tolerance in plants. Front Plant Sci. 2020;11:591911. Islam MJ, Uddin MJ, Hossain MA, Henry R, Begum MK, Sohel MAT, et al. Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet ( Beta vulgaris L). PLoS ONE. 2022;17(1):e0262099. Bahreininejad B, Razmjou J, Mirza M. Influence of water stress on morpho-physiological and phytochemical traits in Thymus daenensis . 2013. Baher ZF, Mirza M, Ghorbanli M, Bagher Rezaii M. The influence of water stress on plant height, herbal and essential oil yield and composition in Satureja hortensis L. Flavour Fragr J. 2002;17(4):275–7. Farooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA. Plant drought stress: effects, mechanisms and management. Sustainable agriculture. Springer; 2009. pp. 153–88. Alavi-Samani SM, Kachouei MA, Pirbalouti AG. Growth, yield, chemical composition, and antioxidant activity of essential oils from two thyme species under foliar application of jasmonic acid and water deficit conditions. Hortic Environ Biotechnol. 2015;56:411–20. Alla Sharafi G, Changizi M, Rafiee M, Gomarian M, Khagani S. Investigating the effect of drought stress and vermicompost biofertilizer on morphological and biochemical characteristics of Thymus vulgaris L. Arch Pharm Pr. 2019;10(3):137–45. Ben Mrid R, Ennoury A, Roussi Z, Naboulsi I, Benmrid B, Kchikich A, et al. Thymoquinone alleviates cadmium induced stress in germinated lens culinaris seeds by reducing oxidative stress and increasing antioxidative activities. Life. 2022;12(11):1779. Leong X-F, Choy KW, Alias A. Anti-Inflammatory Effects of Thymoquinone in Atherosclerosis: A Mini Review. Front Pharmacol. 2021;12:758929. Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM. Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem Funct. 2002;20(2):143–51. Gill SS, Tuteja N. Polyamines and abiotic stress tolerance in plants. Plant Signal \& Behav. 2010;5(1):26–33. Hidalgo-Castellanos J, Duque AS, Burgueno A, Herrera-Cervera JA, Fevereiro P, Lopez-Gomez M. Overexpression of the arginine decarboxylase gene promotes the symbiotic interaction Medicago truncatula-Sinorhizobium meliloti and induces the accumulation of proline and spermine in nodules under salt stress conditions. J Plant Physiol. 2019;241:153034. Galiba G, Kocsy G, Kaur-Sawhney R, Sutka J, Galston AW. Chromosomal localization of osmotic and salt stress-induced differential alterations in polyamine content in wheat. Plant Sci. 1993;92(2):203–11. Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, et al. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta. 2010;231:1237–49. Tabur S, Ozmen S, Oney-Birol S. Promoter role of putrescine for molecular and biochemical processes under drought stress in barley. Sci Rep. 2024;14(1):19202. Hura T, Dziurka Michałand Hura K, Ostrowska A, Dziurka K. Free and cell wall-bound polyamines under long-term water stress applied at different growth stages of $ \times $ Triticosecale Wittm. PLoS ONE. 2015;10(8):e0135002. HU B, NIU M, WANG Q, LI C, LIU H. Relationship between osmotic stress and polyamine levels in leaves of soybean seedlings. J Plant Nutr Fertil. 2006;12(6):881–6. Vondráková Z, Eliášová K, Vágner M, Martincová O, Cvikrová M. Exogenous putrescine affects endogenous polyamine levels and the development of Picea abies somatic embryos. Plant Growth Regul. 2015;75:405–14. Zhao J, Wang X, Pan X, Jiang Q, Xi Z. Exogenous putrescine alleviates drought stress by altering reactive oxygen species scavenging and biosynthesis of polyamines in the seedlings of Cabernet Sauvignon. Front Plant Sci. 2021;12:767992. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6523120","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454046050,"identity":"f88bb0c0-e9a7-43bb-9268-9346d2c721ee","order_by":0,"name":"Ensiyeh Shahroudi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Ensiyeh","middleName":"","lastName":"Shahroudi","suffix":""},{"id":454046051,"identity":"772c57cd-7940-4c5a-a8b1-b67d43e45a80","order_by":1,"name":"Fatemeh Zarinkamar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYJCCA0AIBMxAkg0uKEGMFrYE4rUwQLTwGCBrwQ3k288+PFxwxi6af/aZrxs+lNnl8/MfYPzwg8EiH5cWxp50g8MzbiTnzjiXu+3mjHPJljNnJDBL9jBIWDbg0MLMkMZwmOcDc27DGd5tt3nbmA0MbjAwSAP9YoDLFjb+ZyAt9bnzz/A8A2qpN7A/f4D5Nz4tPBIgW24czt1whocNqOWwgQFDAhteWyQkQLacOZ678QybGdAvxw0kbiS2WfYY4NYi35/G/JnnWHXuvDPMz258KKs24O8/fPjGj4o6nFqwAcYGBgaSNIyCUTAKRsEoQAcAz4pVaNG0s+8AAAAASUVORK5CYII=","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Zarinkamar","suffix":""},{"id":454046052,"identity":"8f61a70b-23cb-4a1c-a7fd-c1af39f7f21a","order_by":2,"name":"Bahram M. soltani","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Bahram","middleName":"M.","lastName":"soltani","suffix":""}],"badges":[],"createdAt":"2025-04-24 18:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6523120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6523120/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07257-4","type":"published","date":"2025-09-30T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82625887,"identity":"376943a4-94a8-46a5-b4e9-383373b0e1a2","added_by":"auto","created_at":"2025-05-13 13:01:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23179,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of drought stress and Put on the content of free Put (a) and free Spd (b) in \u003cem\u003eT. daenensis\u003c/em\u003e. The data are the mean of three replicates ± SE. Different letters show statistically significant differences at \u003cem\u003ep\u003c/em\u003e ≤ 0.05 level.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6523120/v1/8ce1c55ca685ac1f7b7698bd.png"},{"id":92884433,"identity":"a7a2850a-f5a9-4b98-8c70-97aa990d35ef","added_by":"auto","created_at":"2025-10-06 16:12:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":986551,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6523120/v1/563d593c-e3b7-498e-bfe0-2887f5aedabf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Impact of Putrescine on essential oil composition, free polyamines content and expression of related genes (DXR and TPS2) in Thymus daenensis under drought stress","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eT. daenensis\u003c/em\u003e is a species of thyme native to Iran (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It's known for its medicinal properties and is widely used in traditional medicine (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The essential oil of \u003cem\u003eT. daenensis\u003c/em\u003e is rich in compounds like thymol, carvacrol, \u003cem\u003ep\u003c/em\u003e-cymene, and \u003cem\u003eβ\u003c/em\u003e-caryophyllene, which have antioxidant, antibacterial, and antifungal properties (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Thymol and carvacrol exhibit strong antioxidant properties, helping to neutralize reactive oxygen species (ROS) generated during stresses. These compounds reduce oxidative damage to cellular components by stabilizing cell membranes, protecting them from damage in relation to drought-induced dehydration (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Thymol and carvacrol can influence stomatal behavior, promoting stomatal closure to reduce water loss and improve water-use efficiency (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Drought stress can lead to increase in the production of some secondary metabolites. This enhanced production helps the plant cope with stress and defend against pathogens (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Thymol and carvacrol are phenolic monoterpenes, and their production is primarily driven by the methyl erythritol phosphate (MEP) pathway (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The pathway begins with the generation of dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IPP), which are key building blocks for terpenes biosynthesis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). DMADP and IPP are converted into 1-deoxy-D-xylulose-5-phosphate (DXP) through a series of enzymatic reactions. DXP is then transformed into 2C-methyl-D-erythritol-4-phosphate (MEP) via 1-deoxy-D-xylulose-5-phosphate reductoisomerase (\u003cem\u003eDXR\u003c/em\u003e) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). MEP is further processed to produce isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMADP), which are used to synthesize monoterpenes (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). IPP and DMADP are converted into geranyl diphosphate (GPP), which is then cyclized and oxidized to form monoterpene compounds such as thymol and carvacrol by Gamma-terpinene synthase (\u003cem\u003eTPS2\u003c/em\u003e) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The enzymes involved in this pathway, such as \u003cem\u003eDXR\u003c/em\u003e and \u003cem\u003eTPS2\u003c/em\u003e, play crucial roles in the monoterpene biosynthesis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePAs are small aliphatic nitrogen-containing compounds characterized by the presence of two or more amino groups (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These molecules are widely distributed across all living organisms, including both eukaryotic and prokaryotic cells (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The most prevalent forms of PAs include the diamine Put, the triamine spermidine (Spd), and the tetraamine spermine (Spm) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Put is considered the central product in the biosynthetic pathway of PAs, serving as the primary precursor for the synthesis of both Spd and Spm (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have indicated that the application of exogenous PAs can improve plant resistance to drought stress (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These compounds have been found to stimulate the production of secondary metabolites, including alkaloids, phenolic compounds, and terpenoids, which are essential for plant defense and overall health (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Additionally, it has been shown that optimal foliar application of Put can activate key physiological processes, promoting the synthesis of osmotic regulators such as free amino acids, soluble sugars, and proline. This mechanism may help mitigate the negative impacts of drought on plant biomass, thus improving both the quality and quantity of specific bioactive compounds (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePAs play a dual role in regulating oxidative stress in plants (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). On one hand, they enhance the activity of various antioxidant enzymes, thereby helping to mitigate oxidative stress induced by environmental factors (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). On the other hand, PAs can serve as a source of reactive oxygen species (ROS), as their catabolism generates potent oxidants such as hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and acrolein, which may contribute to cellular damage under stress conditions. However, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e also functions as a signaling molecule, initiating the stress signal transduction cascade and activating antioxidant defense mechanisms. Consequently, PAs act as key regulators of redox homeostasis, balancing both protective and potentially harmful effects in response to oxidative stress.\u003c/p\u003e \u003cp\u003eWe hypothesized that the application of Put alleviates drought stress in \u003cem\u003eT. daenensis\u003c/em\u003e by enhancing the biosynthesis and accumulation of some compounds present in the essential oil, regulating the expression of key biosynthetic genes (\u003cem\u003eDXR\u003c/em\u003e and \u003cem\u003eTPS2\u003c/em\u003e) and increasing PAs content. This effect is expected to occur through the modulation of stress-responsive metabolic and genetic pathways, leading to improved drought tolerance and enhanced secondary metabolite production in the plant.\u003c/p\u003e \u003cp\u003eIn essence, our hypothesis suggests that Put plays a protective and stimulatory role in plants under drought conditions, likely involving the coordinated regulation of secondary metabolite biosynthesis, PA accumulation, and specific gene expression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and treatments\u003c/h2\u003e \u003cp\u003eTo conduct this study, \u003cem\u003eT. daenensis\u003c/em\u003e seeds were sourced from the Isfahan Agricultural and Natural Resources Research and Educational Center in Iran. The seeds were sown in individual plastic pots filled with perlite. The pots were kept in a phytotron chamber under light conditions of 16 hours of light and 8 hours of darkness, with a temperature of 25\u0026deg;C and light intensity of 1200\u0026ndash;1400 lux (17\u0026ndash;20 \u0026micro;mol photons/m\u0026sup2;/s) for six weeks. During this time, each pot was irrigated uniformly with a half-strength Hoagland solution.\u003c/p\u003e \u003cp\u003eAfter this initial growth period, drought stress was induced using a polyethylene glycol (PEG) solution (15% W/V), applied with and without Put at a concentration of 0.2 mM as described by (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). 100 mL of the PEG-containing half-strength Hoagland solution was added to each pot, while 10 mL of Put solution was uniformly sprayed onto the plants using an atomizer on alternate days.\u003c/p\u003e \u003cp\u003eEach experimental treatment was performed in triplicate to ensure data reliability. At the end of the three-week treatment period, shoots were collected from each condition and a portion of the them were dried, while the remaining ones were stored \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTotal RNA extraction and cDNA synthesis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated utilizing the Riboex reagent according to the protocol provided by the manufacturer. The concentration and purity of the extracted RNA were measured using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and verified by agarose gel electrophoresis. Samples exhibiting an A260/A280 ratio between 1.8 and 2.1 and an A260/A230 ratio above 1.7 were selected for downstream complementary DNA (cDNA) synthesis.\u003c/p\u003e \u003cp\u003ecDNA was synthesized from the purified RNA using reverse transcription kits obtained from Thermo Fisher (USA), adhering strictly to the provided instructions. Quantitative real-time PCR (RT-qPCR) was performed to evaluate the expression of \u003cem\u003eDXR\u003c/em\u003e and \u003cem\u003eTPS2\u003c/em\u003e genes, employing SYBR Green PCR Master Mix (2\u0026times;) (Yekta Tajhiz Azma, Tehran, Iran). The amplification protocol involved an initial denaturation at 95\u0026deg;C for 5 minutes, followed by 40 cycles comprising 10 seconds at 95\u0026deg;C for denaturation, 10 seconds at 60\u0026deg;C for annealing, and 15 seconds at 72\u0026deg;C for extension. Reactions were run on an Applied Biosystems\u0026reg; StepOne\u0026trade; instrument (Thermo Fisher, USA), and data analysis was conducted using the StepOne Software v2.3. Gene expression levels were normalized to \u003cem\u003eGAPDH\u003c/em\u003e and analyzed using the 2^\u0026ndash;ΔΔCt method described by Livak and Schmittgen (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). All assays were carried out with two independent biological replicates and two technical replicates per sample.\u003c/p\u003e\n\u003ch3\u003ePrimers designing\u003c/h3\u003e\n\u003cp\u003eFor primer designing, the sequence of target genes was first examined on the NCBI website (gov.nih.nlm.ncbi.www). The sequence for the \u003cem\u003eGamma terpinene synthase\u003c/em\u003e (\u003cem\u003eTPS2\u003c/em\u003e) gene was found for the \u003cem\u003eT. daenensis\u003c/em\u003e species. However, no registered sequences for the \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eDXR\u003c/em\u003e genes of this species were available. Therefore, gene sequences from a closely related species, \u003cem\u003eT. vulgaris\u003c/em\u003e, were used. The accuracy of these sequences was confirmed by comparison with the gene bank, and primers for Real-Time PCR (RT-qPCR) were then designed based on these sequences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecifications of primers used for RT-qPCR (Real time PCR)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmplicone size (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccession no.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward:5ʹGTGCTTCCAGCTTTGAACG-3ʹ\u003c/p\u003e \u003cp\u003eRiverse: 5ʹGTTCTCTGACTCCTCCTTGATG-3ʹ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;MF373628.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDXR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: 5ʹ-GTGCTAGCTCAGTTAGGA TGG-3ʹ\u003c/p\u003e \u003cp\u003eRiverse: 5ʹ-TTAGATCGACGTTGCAGAGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;KY621335.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTPS2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward: 5ʹ-CTTACAAGGCGAGGA AGG ACA-3ʹ\u003c/p\u003e \u003cp\u003eRiverse: 5ʹ-CACAAATGGGAGTTTCTCGG-3ʹ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;MH686193.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMeasurement of PA content using HPLC\u003c/h3\u003e\n\u003cp\u003ePAs were extracted following the method of Sharma \u0026amp; Rajam (1995). 0.2 g of samples was homogenized with 1 mL of 2% perchloric acid (PCA) and centrifuged at 4\u0026deg;C for 20 minutes. Then, 150 \u0026micro;L of the supernatant was mixed with 200 \u0026micro;L of saturated sodium carbonate and 500 \u0026micro;L of dansyl chloride solution (5 mg/mL) for dansylation. The mixture was incubated at 60\u0026deg;C in the dark for 1 hour. Afterward, 200 \u0026micro;L of proline solution (0.1 g/mL) was added, and the solution was kept under the same conditions for an additional 30 minutes. The dansylated PAs were extracted by vortexing with 500 \u0026micro;L of toluene, and the upper phase containing the PAs was collected for subsequent analysis. PAs quantification was performed using an ODS18-C5 column. The mobile phase consisted of acetonitrile and acidified water (deionized water with 0.01% acetic acid) applied using a standard gradient system. The column used was an ODS18-C3 type with a length of 250 mm and a diameter of 4.6 mm. PAs absorption was detected using a UV detector at a wavelength of 254 nm. The quantification of PAs was performed based on the chromatogram, using their respective standard curves (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGC.MS\u003c/h3\u003e\n\u003cp\u003e20 mg portion of powdered plant dry tissue was placed in 20 mL vials. Static headspace analysis was performed using an A7697 Headspace Sampler (Agilent Technologies). The headspace oven temperature was maintained at 80\u0026deg;C for 30 minutes to allow volatile compounds to evaporate and reach equilibrium between the headspace and the sample in the vial. Subsequently, a specialized syringe was used to extract 1 mL of the headspace, which was then injected into a gas chromatography-mass spectrometry (GC-MS) system for analysis.\u003c/p\u003e \u003cp\u003eAn Agilent 7890 gas chromatograph, coupled with an HP-5MS column (30 m length, 0.25 mm internal diameter, and 250 \u0026micro;m film thickness), and an ELCD 5320 detector, was employed for the analysis. The temperature program for the oven was as follows: an initial ramp from 60\u0026deg;C to 210\u0026deg;C at a rate of 3\u0026deg;C/min, followed by a temperature increase to 240\u0026deg;C at 20\u0026deg;C/min, and a subsequent hold at 240\u0026deg;C for 8.5 minutes. The total run time was 60 minutes, with the electron ionization energy set to 70 eV. Helium was used as the carrier gas with a flow rate of 1 mL/min. Data analysis was conducted using ChemStation software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were analyzed using analysis of variance (ANOVA) based on a completely randomized design (CRD) with three replications. Duncan\u0026rsquo;s test was applied to determine statistically significant differences between treatment means at a significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eRelative Content of Essential Oil Compounds and Expression of\u003c/b\u003e \u003cb\u003eDXR\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eTPS2\u003c/b\u003e \u003cb\u003eGenes\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe impact of drought stress on essential oil content in plants varies depending on the plant species, the intensity and duration of stress, and the growth stage (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Some studies have shown that drought stress can enhance essential oil content by stimulating the biosynthesis of terpenoids, phenylpropanoids, and other aromatic compounds (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Conversely, other studies have indicated that drought stress can reduce essential oil content by limiting biomass production and the availability of precursors (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Thus, the optimal irrigation level for maximizing essential oil content depends on the plant species and environmental conditions (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA study on \u003cem\u003eT. daenensis\u003c/em\u003e demonstrated that Put increased the content of thymol and other secondary metabolites such as carvacrol, \u003cem\u003eα\u003c/em\u003e and \u003cem\u003eγ-\u003c/em\u003eterpinene, \u003cem\u003ep\u003c/em\u003e-cymene, and \u003cem\u003eβ\u003c/em\u003e-caryophyllene (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Moreover, it increased the content of terpenoids, especially mono- and sesquiterpenes, in tea plants under saline stress (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Another study found that Put reduced terpenoid content in thyme under drought stress but increased the phenolic compound content (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These findings suggest that Put regulates the biosynthesis of various secondary metabolites in plants, depending on environmental conditions and genetic factors.\u003c/p\u003e \u003cp\u003eGenerally, the mechanism of PAs effects under drought stress is not fully understood but may involve regulation of enzyme gene expression and their activity, transcription factors, and signaling pathways (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In this study, the thymol content increased by 4.4% in drought-stressed plants compared to controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similar results were previously observed in garden thyme (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and oregano (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Research on \u003cem\u003eT. daenensis\u003c/em\u003e subsp. \u003cem\u003edaenensis\u003c/em\u003e showed that drought stress (20% field capacity) increased thymol content by 17.5% (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Additionally, drought stress (50% field capacity) increased thymol content by 13.6% in the first growth season and 9.8% in the second growth season, compared to controls (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In addition to drought stress at 40% field capacity, thymol content increased by 10.6% in the first year and 7.8% in the second year in \u003cem\u003eT. daenensis\u003c/em\u003e (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). These results suggest that drought stress enhances thymol biosynthesis as a defensive mechanism against oxidative stress in \u003cem\u003eT. daenensis\u003c/em\u003e. However, the optimal level and duration of drought stress depend on plant genotype, climate, and soil conditions (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The ratio of thymol to other compounds in the essential oil affects the quality of cosmetic, food, and pharmaceutical products derived from this plant (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), Therefore, it can be suggested that the quality of \u003cem\u003eT. daenensis\u003c/em\u003e essential oil may improve under drought stress.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of drought stress and Put on the relative content of compounds in the essential oil of \u003cem\u003eT. daenensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003esubstance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eKI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eRelative content of substance (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePut\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePEG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePut\u0026thinsp;+\u0026thinsp;PEG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.53\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45.49\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.37\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36.27\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e41.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarvacrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.36\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.50\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.25\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eγ\u003c/em\u003e-Terpinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1050\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.34\u003csup\u003ed\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.71\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.67\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.00\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-cymene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.60\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.18\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.87\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.87\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e13.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eα\u003c/em\u003e-Thujene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.42\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.50\u003csup\u003ec\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.07\u003csup\u003eb\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.30\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCamphene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.57\u003csup\u003ea\u003c/sup\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.45 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.66 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eβ\u003c/em\u003e-Pinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.66 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.69 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.69 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eβ\u003c/em\u003e -Myrcene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.50 \u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.82 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.49 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eα\u003c/em\u003e -Terpinen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.50 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.44 \u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.14 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eβ\u003c/em\u003e -Phellandrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eα\u003c/em\u003e \u0026ndash; Phellandrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndo-Borneol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.35 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.48 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.14 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThymol methyl ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25 \u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.37 \u003csup\u003ebc\u003c/sup\u003e\u0026plusmn;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.46 \u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarvacrol methyl ether\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.63 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.46 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.88 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.35 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThymoquinone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.95 \u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.57 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.94 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33 \u003csup\u003ed\u003c/sup\u003e\u0026plusmn;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCaryophyllene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.50 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.72 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.73 \u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.20 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAromandendrene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.18 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.19 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHumulene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.10 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.09 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eβ\u003c/em\u003e -Bisabolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.32 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.44 \u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.43 \u003csup\u003eab\u003c/sup\u003e\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(Z)- \u003cem\u003eα\u003c/em\u003e -Bisabolene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.07 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.34 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.11 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.39 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eα\u003c/em\u003e -Pinene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.25 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.73 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEucalyptol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.39 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.57 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.63 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(+)-2-Carene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e995\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003end\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.25 \u003csup\u003ea\u003c/sup\u003e\u0026plusmn;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.53 \u003csup\u003ebc\u003c/sup\u003e\u0026plusmn;0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.91 \u003csup\u003ec\u003c/sup\u003e\u0026plusmn;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.22 \u003csup\u003eb\u003c/sup\u003e\u0026plusmn;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe data are the mean of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Different letters show statistically significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 level.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of drought stress and Put on the relative expression of the \u003cem\u003eDXR\u003c/em\u003e and \u003cem\u003eTPS2\u003c/em\u003e gene in \u003cem\u003eT. daenensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDXR\u003c/em\u003e expression\u003c/p\u003e \u003cp\u003e(fold change)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTPS2\u003c/em\u003e expression\u003c/p\u003e \u003cp\u003e(fold change)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePut (0.2 mM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.31\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePEG (15%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.34\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u003csup\u003eC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePut\u0026thinsp;+\u0026thinsp;PEG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe data are the mean of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Different letters show statistically significant differences at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 level.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Put treatment increased thymol content by 9.5%. However, in plants under drought stress, Put caused a 16.37% reduction in thymol content (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). One possible explanation for this reduction is that Put regulates the biosynthesis of various secondary metabolites based on genetic factors and environmental conditions (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Consequently, Put may reduce thymol content while increasing the levels of other secondary metabolites that could play a more protective role under stress conditions (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on our results, drought stress also reduced carvacrol content by 6.25% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Previous studies reported a decline in carvacrol content in \u003cem\u003eT. daenensis\u003c/em\u003e (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), garden thyme (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and Himalayan thyme (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) under drought stress. In contrast, it is found that carvacrol content increased under mild drought stress but decreased under severe stress in \u003cem\u003eSatureja hortensis\u003c/em\u003e (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Under non-stress and stress conditions, Put reduced the carvacrol content by 15% and 16.6%, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Previous studies reported a positive effect of Put on carvacrol content; for instance, Put increased carvacrol content by 2.10% in oregano. A potential explanation for the reduction in carvacrol content with Put treatment is the conversion of carvacrol to thymoquinone, as Put increased thymoquinone (TQ) content by 83% in non-stressed plants.\u003c/p\u003e \u003cp\u003eThe content of \u003cem\u003ep\u003c/em\u003e-cymene, a precursor for thymol and carvacrol, showed a significant decrease (43.14%) under drought stress (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similar results were observed in \u003cem\u003eT. daenensis\u003c/em\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) and \u003cem\u003eSatureja hortensis\u003c/em\u003e (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). This negative effect could be due to the inhibitory role of drought stress on the monoterpene biosynthesis pathway and reduced activity of related enzymes (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Also, our results indicated that Put reduced \u003cem\u003ep\u003c/em\u003e-cymene content by 2.6% under non-stress conditions while increased it\u0026rsquo;s content by 56% under drought stress (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe expression of \u003cem\u003eTPS2\u003c/em\u003e, the gene encoding the enzyme involved in \u003cem\u003eγ\u003c/em\u003e-terpinene biosynthesis, increased 1.5-fold under drought stress (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Studies have shown that \u003cem\u003eγ\u003c/em\u003e-terpinene content increases under mild to moderate drought stress in thyme species (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), while it decreases under severe stress, as observed in garden thyme (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) and \u003cem\u003eT. daenensis\u003c/em\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The discrepancy in results may be related to the plant's adaptive mechanisms, such as changes in proline content, potassium levels, and antioxidant activity, in response to drought (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Put treatment increased \u003cem\u003eγ\u003c/em\u003e-terpinene content by 89.6%, (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with a 23.3-fold increase in \u003cem\u003eTPS2\u003c/em\u003e gene expression compared to controls (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under drought stress, Put enhanced \u003cem\u003eγ\u003c/em\u003e-terpinene content by 28.5-fold and \u003cem\u003eTPS2\u003c/em\u003e gene expression by 2.7-fold (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTQ, an important compound in the essential oil of \u003cem\u003eT. daenensis\u003c/em\u003e, is derived from thymol and carvacrol. Drought stress increased TQ content by 50.7% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Mild drought stress (40% field capacity) increased TQ content, while severe drought stress (20% field capacity) decreased it (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). TQ has been suggested to enhance the antioxidant and antimicrobial activities of thyme essential oil. Studies indicate that TQ mitigates drought stress by modulating growth regulators like abscisic acid, salicylic acid, and jasmonic acid (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e).TQ also boosts the activity of enzymes like superoxide dismutase, catalase, and ascorbate peroxidase (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e), as well as non-enzymatic antioxidants such as ascorbic acid, glutathione, and phenolic compounds. Additionally, TQ improves relative water content (RWC), water use efficiency (WUE), and proline accumulation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e\u003cb\u003eFree PAs Content\u003c/b\u003e\u003c/div\u003e \u003cp\u003eDuring various stress conditions, the accumulation of PAs contributes to plant survival and stress tolerance. The ability of PAs to mitigate stress effects is attributed to their antioxidant properties (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) and their role in maintaining cellular turgor pressure (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). PAs in plants are synthesized through the enzymatic activities of ornithine decarboxylase and arginine decarboxylase. Under stress conditions, the biosynthesis of these compounds is increased through the activity of arginine decarboxylase (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNumerous studies have been reported that PAs have a modulating function in plants exposed to drought stress (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Osmotic stress induced by mannitol has been shown to increase the levels of Put, Spd, and Spd in wheat plants (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Research findings indicate that under drought stress, the activity of ornithine decarboxylase and arginine decarboxylase, as well as the content of PAs, is higher in drought-tolerant species compared to drought-sensitive ones (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This underscores the role of PAs in enhancing plant resistance to stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDrought stress decreased the content of endogenous Put by 26.3% and had no effect on Spd content. A study on barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.) seedlings reported that 21-day drought stress resulted in an 80% reduction in endogenous Put levels. However, the same stress conditions led to increases in Spd (50%) level.\u003c/p\u003e \u003cp\u003ePut treatment increased the free Put content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.a) by 55% and free Spd content (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.b) by 23.1% in plants under drought stress. The application of exogenous putrescine under drought conditions was found to mitigate some of the adverse effects of drought stress (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Similarly, researchers observed that drought stress led to a decrease in free Spd levels at all developmental stages in both traditional and semi-dwarf triticale (\u0026times;\u003cem\u003eTriticosecale Wittm\u003c/em\u003e.) cultivars. Additionally, a reduction in free Put was noted in the traditional cultivar under drought conditions (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In another study, the effects of osmotic stress induced by PEG on polyamine content in soybean (\u003cem\u003eGlycine max\u003c/em\u003e L.) leaves were examined. The findings indicated that drought-tolerant soybean cultivars exhibited higher increases in free Spd and Spm levels, while the drought-sensitive cultivars showed a higher increase in free putrescine under stress conditions. This suggests a complex relationship between polyamine levels and drought tolerance in soybean (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExogenous application of Put significantly influences the endogenous levels of PAs, including Put itself and Spd, in plants. The effects observed can vary depending on the plant species, developmental stage, and environmental conditions (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). These changes are mediated through the regulation of genes associated with PA metabolism, highlighting the intricate balance plants maintain in PA homeostasis. Applying Put externally often leads to an increase in the plant's internal Put concentration. For instance, in \u003cem\u003ePicea abies\u003c/em\u003e somatic embryos, treatment with exogenous Put at concentrations of 10, 100, or 500 \u0026micro;M resulted in a significant, concentration-dependent rise in endogenous Put levels. This suggests that externally supplied Put can be absorbed and utilized by plant tissues, thereby elevating internal Put (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConversely, research on \u003cem\u003eVitis vinifera\u003c/em\u003e (grapevine) seedlings under drought stress demonstrated that exogenous Put application not only increased endogenous Put content but also influenced Spd levels. Specifically, certain treatments led to a significant rise in Spd content, suggesting that exogenous Put can serve as a precursor for the biosynthesis of higher PAs like Spd, especially under stress conditions (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights the significant role of Put in mitigating drought stress in \u003cem\u003eT. daenensis\u003c/em\u003e by modulating gene expression, PAs accumulation, and secondary metabolite composition. The findings highlight that exogenous Put application significantly increases endogenous Put and Spd levels under drought conditions, suggesting its involvement in osmotic adjustment and oxidative stress regulation.\u003c/p\u003e \u003cp\u003eAt the molecular level, Put treatment upregulated \u003cem\u003eTPS2\u003c/em\u003e while downregulating \u003cem\u003eDXR\u003c/em\u003e expression in drought-stressed plants, indicating its influence on the terpenoid biosynthetic pathway. These genetic modifications were accompanied by a decrease in thymol and carvacrol content, alongside an increase in their precursors, \u003cem\u003eγ\u003c/em\u003e-terpinene and \u003cem\u003ep\u003c/em\u003e-cymene. This shift in essential oil composition suggests that Put may regulate metabolic fluxes within the plant\u0026rsquo;s secondary metabolism in response to drought stress.\u003c/p\u003e \u003cp\u003eOverall, these results underscore the potential of PAs, particularly Put, as biostimulants for improving drought tolerance and metabolic regulation in medicinal plants. Further investigations are needed to assess the long-term physiological effects and potential agronomic applications of Put in \u003cem\u003eT. daenensis\u003c/em\u003e cultivation under field conditions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMAPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDimethylallyl pyrophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDXP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1-deoxy-D-xylulose 5-phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eDXR\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1-deoxy-D-xylulose 5-phosphate reductoisomerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eGAPDH\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGeranyl pyrophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH₂O₂\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydrogen peroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIsopentenyl pyrophosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMethylerythritol phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolyamine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolyethylene glycol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePut\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePutrescine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative real-time PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSpd\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpermidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSpm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpermine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eT\u003c/em\u003e. \u003cem\u003edaenensis\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eThymus daenensis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eTPS2\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTerpene synthase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to express my sincere gratitude to the University of Tarbiat Modares for providing me with the necessary resources and support throughout the course of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnsiyeh Shahroudi designed and performed the experiments, collected and analyzed the data, and wrote the manuscript. Dr. Fatemeh Zarinkamar supervised the physiological experiments conducted in her laboratory. Dr. Bahram M. Soltani supervised the molecular experiments conducted in his laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no external funding for this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve any human participants or animals. \u003cem\u003eThymus daenensis\u003c/em\u003e seeds were sourced from the Isfahan Agricultural and Natural Resources Research and Educational Center (Iran), and the plants were cultivated and used in accordance with institutional, national, and international guidelines. Therefore, no ethical approval or consent to participate was required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlizadeh A, Alizadeh O, Amari G, Zare M. Essential oil composition, total phenolic content, antioxidant activity and antifungal properties of Iranian \u003cem\u003eThymus daenensis\u003c/em\u003e subsp. daenensis Celak. as in influenced by ontogenetical variation. J Essent Oil Bear Plants. 2013;16(1):59\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhasemi Pirbalouti A, Rahmani Samani M, Hashemi M, Zeinali H. 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Int Agrophysics. 2009;23(3):269\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKachoie MA, Pirbalouti AG, Hamedi B, Borojeni HAR, Pishkar GR. Effect of drought stress on antibacterial activity of \u003cem\u003eThymus daenensis\u003c/em\u003e subsp. \u003cem\u003edaenensis\u003c/em\u003e Celak. J Med Plants Res. 2013;7(36):2923\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBistgani ZE, Siadat SA, Bakhshandeh A, Pirbalouti AG, Hashemi M. Interactive effects of drought stress and chitosan application on physiological characteristics and essential oil yield of \u003cem\u003eThymus daenensis\u003c/em\u003e Celak. Crop J. 2017;5(5):407\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweiger AH, Zimmermann T, Poll C, Marhan S, Leyrer V, Berauer BJ. The need to decipher plant drought stress along the soil\u0026ndash;plant\u0026ndash;atmosphere continuum. Oikos. 2023;2023(9):e10136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetchamo W, Xu HL, Gosselin A. Photosynthetic potential of Thymus vulgaris selections under two light regimes and three soil water levels. Sci Hortic (Amsterdam). 1995;62(1\u0026ndash;2):89\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Y, Dias MC, Freitas H. Drought and salinity stress responses and microbe-induced tolerance in plants. Front Plant Sci. 2020;11:591911.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslam MJ, Uddin MJ, Hossain MA, Henry R, Begum MK, Sohel MAT, et al. Exogenous putrescine attenuates the negative impact of drought stress by modulating physio-biochemical traits and gene expression in sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e L). PLoS ONE. 2022;17(1):e0262099.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBahreininejad B, Razmjou J, Mirza M. Influence of water stress on morpho-physiological and phytochemical traits in \u003cem\u003eThymus daenensis\u003c/em\u003e. 2013.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaher ZF, Mirza M, Ghorbanli M, Bagher Rezaii M. The influence of water stress on plant height, herbal and essential oil yield and composition in \u003cem\u003eSatureja hortensis\u003c/em\u003e L. Flavour Fragr J. 2002;17(4):275\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarooq M, Wahid A, Kobayashi N, Fujita D, Basra SMA. Plant drought stress: effects, mechanisms and management. Sustainable agriculture. Springer; 2009. pp. 153\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlavi-Samani SM, Kachouei MA, Pirbalouti AG. Growth, yield, chemical composition, and antioxidant activity of essential oils from two thyme species under foliar application of jasmonic acid and water deficit conditions. Hortic Environ Biotechnol. 2015;56:411\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlla Sharafi G, Changizi M, Rafiee M, Gomarian M, Khagani S. Investigating the effect of drought stress and vermicompost biofertilizer on morphological and biochemical characteristics of \u003cem\u003eThymus vulgaris\u003c/em\u003e L. Arch Pharm Pr. 2019;10(3):137\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBen Mrid R, Ennoury A, Roussi Z, Naboulsi I, Benmrid B, Kchikich A, et al. Thymoquinone alleviates cadmium induced stress in germinated lens culinaris seeds by reducing oxidative stress and increasing antioxidative activities. Life. 2022;12(11):1779.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeong X-F, Choy KW, Alias A. Anti-Inflammatory Effects of Thymoquinone in Atherosclerosis: A Mini Review. 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J Plant Physiol. 2019;241:153034.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaliba G, Kocsy G, Kaur-Sawhney R, Sutka J, Galston AW. Chromosomal localization of osmotic and salt stress-induced differential alterations in polyamine content in wheat. Plant Sci. 1993;92(2):203\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlc\u0026aacute;zar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, et al. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta. 2010;231:1237\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabur S, Ozmen S, Oney-Birol S. Promoter role of putrescine for molecular and biochemical processes under drought stress in barley. Sci Rep. 2024;14(1):19202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHura T, Dziurka Michałand Hura K, Ostrowska A, Dziurka K. 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Exogenous putrescine alleviates drought stress by altering reactive oxygen species scavenging and biosynthesis of polyamines in the seedlings of Cabernet Sauvignon. Front Plant Sci. 2021;12:767992.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Drought stress, Gene expression (DXR, TPS2), Putrescine, Terpenoid biosynthesis, Thymus daenensis","lastPublishedDoi":"10.21203/rs.3.rs-6523120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6523120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground Thymus daenensis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea medicinal plant native to Iran, produces essential oils rich in thymol and carvacrol, known for their strong antioxidant and antimicrobial properties. Exposure to drought conditions affects the production pathways of secondary metabolites, potentially contributing to improved stress tolerance in plants. Polyamines (PAs), particularly putrescine (Put), play a crucial role in mitigating drought stress by regulating stomatal behavior, improving osmotic adjustment, and modulating oxidative stress responses. This study aimed to investigate the effects of exogenous Put on \u003cem\u003eT. daenensis\u003c/em\u003e under drought stress, focusing on gene expression related to terpenoid biosynthesis, secondary metabolite accumulation, and free PA content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix-week-old \u003cem\u003eT. daenensis\u003c/em\u003e plants were subjected to drought stress induced by 15% (w/v) polyethylene glycol (PEG) 6000, with or without foliar application of Put. Gene expression analysis (RT-qPCR), HPLC-based PA quantification, and GC-MS profiling of essential oil composition were performed. Put treatment significantly increased endogenous Put and spermidine content in drought-stressed plants, whereas no significant changes were observed in non-stressed plants. Furthermore, Put application upregulated \u003cem\u003eTPS2\u003c/em\u003e but downregulated \u003cem\u003eDXR\u003c/em\u003e expression in drought-stressed plants. Additionally, the relative contents of \u003cem\u003eγ\u003c/em\u003e-terpinene and \u003cem\u003ep\u003c/em\u003e-cymene—precursors of thymol and carvacrol—increased, while the contents of thymol and carvacrol decreased following Put treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExogenous application of putrescine modulates the expression of key genes in the terpenoid biosynthetic pathway and alters essential oil composition in \u003cem\u003eT. daenensis\u003c/em\u003e under drought stress, potentially contributing to the plant’s adaptive responses.\u003c/p\u003e","manuscriptTitle":"Impact of Putrescine on essential oil composition, free polyamines content and expression of related genes (DXR and TPS2) in Thymus daenensis under drought stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 13:01:31","doi":"10.21203/rs.3.rs-6523120/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-30T23:45:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-28T09:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11461063326792578442868937499484678639","date":"2025-06-18T16:40:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T09:20:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"220182413224903230630571831859706443622","date":"2025-06-16T03:59:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287755156603843111667857483637252210347","date":"2025-06-13T18:32:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"105815932279258861727182254847055643032","date":"2025-06-13T18:21:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13168207904144215679831039073971423563","date":"2025-06-13T17:28:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T08:35:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252067042056136501657912311142628210371","date":"2025-05-21T18:28:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"12139058802531611247759066040605470133","date":"2025-05-09T00:14:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-08T16:02:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-08T15:34:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-07T07:04:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-07T06:57:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-04-24T18:15:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b0a482a-05d6-4762-ba5a-a11cb94e71a9","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:08:50+00:00","versionOfRecord":{"articleIdentity":"rs-6523120","link":"https://doi.org/10.1186/s12870-025-07257-4","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-09-30 15:56:54","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-05-13 13:01:31","video":"","vorDoi":"10.1186/s12870-025-07257-4","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07257-4","workflowStages":[]},"version":"v1","identity":"rs-6523120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6523120","identity":"rs-6523120","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}